Leaf and fruit size are the crucial fitness characters for plant evolution and the agronomic traits for crop yield and quality improvement in watermelon. However, the underlying genes and molecular mechanisms for regulating fruit size and biomass remain elusive. Here, we identified a Citrullus lanatus UDP-rhamnose (Rha)/UDP-galactose (Gal) transporter ClURGT4, which is localized in the Golgi apparatus and has the dual functions of transporting UDP-Gal and UDP-Rha. Loss-of-function clurgt4 mutants resulted in decreased biomass and reduced Gal, Rha, and galacturonic acid (GalA) in cell wall components. Microscopical analysis showed that ClURGT4 promoted leaf and fruit size by modulating cell expansion. Proteomic analysis revealed that several cell wall metabolism-related proteins were changed in clurgt4 mutants. Moreover, protein glycosylation was changed in the mutants, and several of the differentially glycosylated proteins were related to cell wall metabolism. These findings elucidated that ClURGT4 might control leaf and fruit size by affecting cell wall metabolism and provided a novel case for comprehensively revealing the regulatory network of watermelon biomass.

Watermelon (Citrullus lanatus), an important member of the Cucurbitaceae family, has become one of the most popular economic crops in the world, and understanding its fruit development and ripening has always been a hot topic. Although DNA methylation is known to play an essential role in fruit ripening, its contribution to watermelon ripening remains unknown. Here, we found a global and conspicuous hypermethylation pattern during watermelon fruit development and ripening, which is opposite to the hypomethylation found in tomato and strawberry. Application of the DNA methylation inhibitor 5-azacytidine (5-Aza) delayed fruit ripening, confirming that the positive role of hypermethylation in this process. The hypermethylation mainly occurred in CHG and CHH types, and gene-body hypermethylation strongly correlated with the expression of genes that related to sugar metabolism and ABA (abscisic acid) response. We also found an overall hypermethylation pattern in domesticated watermelon. ClROS1 (Repressor Of Silencing 1), a gene encoding the DNA demethylase, was under selection; its expression was negatively correlated with DNA methylation levels, suggesting that it contributes to the hypermethylation during ripening. Collectively, our research illuminates the genome-wide DNA methylation dynamics during watermelon ripening and provides an invaluable genetic resource that will greatly benefit the molecular breeding and quality improvement of this important crop.

Pangenomes are increasingly important for harnessing crop genetic diversity, yet their resolution and utility are often limited by insufficient sampling of high-quality genome assemblies. Here we present a population-level watermelon super-pangenome constructed from 138 reference-grade assemblies, including 135 newly generated genomes representing all seven species. This super-pangenome captures approximately 1 million structural variants (SVs), enabling accurate variant genotyping across 914 accessions. Broader sampling within the pangenome provides insights into watermelon genome evolution and the origin of cultivated watermelon. Incorporating SVs into genome-wide association studies improves mapping resolution and reveals a copy number variant upstream of ClFCI1 that regulates flesh color intensity in a dosage-dependent manner. Leveraging this comprehensive variation map, we developed high-accuracy genomic prediction models for 18 agronomic traits. Together, these findings and genomic resources establish a foundation for dissecting complex traits and accelerating precision breeding in watermelon, while offering a valuable model for SV-resolved pangenomics in crops.

Heterosis, characterized by enhanced resistance and yield, has been widely utilized in watermelon breeding. However, our understanding of the regulatory mechanisms underlying male-sterile phenotypes in watermelon remains limited. Here, we determined that the miR159a targets ClMYB33 to regulate anther dehiscence, leading to male sterility in watermelon. Both overexpression of Cl-miR159a (OE-miR159a) and knockout of ClMYB33 (clmyb33) in watermelon suppressed the degradation of septum and stomium tissues, thereby impairing anther dehiscence and preventing successful pollen release. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, ClPG1 and ClQRT2 were identified as downstream target genes of ClMYB33; both were positively regulated by ClMYB33. Both ClPG1 and ClQRT2 exhibited polygalacturonase (PG) activity in vivo. The knockout of ClQRT2 led to reduced PG activity and a failure in anther dehiscence. Furthermore, the GST-ClQRT2 fusion protein was capable of rescuing the indehiscent anther phenotype observed in both OE-miR159a and clmyb33 plants. Our results reveal a new mechanism by which the miR159a-ClMYB33 module regulates anther dehiscence by mediating PG activity, and provide a new molecular tool to create male sterility in watermelon.

Vegetables are crucial to human diet and health. To ensure sustainable vegetable production, regulatory measures are needed to enhance seed germination, plant growth, and resilience to extreme environmental conditions. Nanomaterials (NMs), owing to their high surface area, nanoscale dimensions, and unique photocatalytic properties, exhibit remarkable biological effects, such as promoting germination and growth, as well as improving stress resistance in crops, offering novel solutions to key challenges in vegetable cultivation. This review summarizes the absorption pathways of NMs in plants, specifically through the leaves and roots of vegetables. Their uptake and translocation occur via passive diffusion, active transport, and endocytosis, with key influencing factors including particle size, chemical composition, surface charge, and surface modifications. We further evaluate the advantages of nanofertilizers and nanopesticides, in vegetable production over their traditional counterparts, focusing on improvements in seed germination rates, seedling vigor, biotic and abiotic stress tolerance, and overall yield and quality. Through this review, we aim to offer comprehensive insights into the application of NMs in vegetable crop production.

The regulation of non-climacteric fruit ripening by the transcription factor NON-RIPENING (NOR) is poorly understood. Here, we identified that the NOR homolog in the non-climacteric fruit watermelon (Citrullus lanatus) was located within the selective sweep and sweetness quantitative trait locus that was selected during domestication from landraces to cultivars. ClNOR knockout substantially delayed fruit ripening, and the fruits of the knockout plants had lower abscisic acid (ABA) levels, lighter colored flesh, and were less sweet compared to wild type. Transcriptome analysis and DNA affinity purification sequencing revealed that ClNOR targeted the Basic Leucine Zipper gene ClbZIP1, which links ClNOR to genes that do not have a ClNOR-binding motif in their promoters, such as the ABA biosynthesis gene, 9-cis-epoxycarotenoid dioxygenase ClNCED1 and the chromoplast phosphate transporter gene ClPHT4;2. The double mutant Clnor Clbzip1 exhibited delayed fruit ripening, lower ABA level, and lighter colored flesh. Its delayed ripening phenotype was stronger than that of the Clbzip1 single mutant. Additionally, the ClNORT,T haplotype in cultivated watermelon resulted in higher ClbZIP1 expression, but ClNORC,T from landraces and ClNORC,G from ancestral watermelon did not. Heterologous ClNORT,T expression rescued the delayed ripening phenotype of the Slnor knockout in tomato (Solanum lycopersicum). This natural variant (564T/C) of ClNOR promoted fruit ripening by enhancing target genes transcription. Overall, these findings will help elucidate the evolutionary mechanisms of nonclimacteric fruit ripening.

Watermelon (Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid (ABA) signaling pathway gene ClSnRK2.3 might influence watermelon fruit ripening. However, the molecular mechanisms are unclear. Here, we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA(4). Furthermore, we determined that the pyrophosphate-dependent phosphofructokinase (ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase (ClGA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA(4). Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein (ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9'-cis-epoxycarotenoid dioxygenase 3 (ClNCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA(4). Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.

Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for watermelon fruit ripening have not been comprehensively studied. In this study, we conducted PacBio single-molecule long-read sequencing to identify genome-wide alternative splicing (AS), alternative polyadenylation (APA) and long non-coding RNAs (lncRNAs) in watermelon fruit. In total, 6,921,295 error-corrected and mapped full-length non-chimeric (FLNC) reads were obtained. Notably, more than 42,285 distinct splicing isoforms were derived from 5,891,183 intron-containing full-length FLNC reads, including a large number of AS events associated with fruit ripening. In addition, we characterized 21,506 polyadenylation sites from 11,611 genes, 8703 of which have APA sites. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that fructose and mannose metabolism, starch and sucrose metabolism and carotenoid biosynthesis were both enriched in genes undergoing AS and APA. These results suggest that post-transcriptional regulation might potentially have a key role in regulation of fruit ripening in watermelon. Taken together, our comprehensive PacBio long-read sequencing results offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of watermelon fruit ripening.

为满足盐城地区市场多元化的需求,丰富本地区的小果型西瓜品种,对引进的5个小型西瓜新品种进行栽培试验.结果表明:京美1K和京美2K生长势较强,果实发育期短,分别为39 d和37 d,且其抗病性较强,瓜皮韧性高,耐贮运性好,果实品质优,中心与边缘可溶性固形物含量较为均匀,中心在12.00％以上,边缘在11.00％以上;京彩1号、京彩3号和京彩6号属于早熟耐裂橙、黄瓤色类型品种,果形稳定,口感脆爽,较耐运输和存放.综上,5个小型西瓜品种生长势强,早熟性好,抗病性、耐贮运性佳,产量较高,适宜在盐城地区根据各品种特性进行秋茬设施栽培.

The Cucurbitaceae (cucurbit) family consists of about 1,000 species in 95 genera, including many economically important and popular fruit and vegetable crops. During the past several years, reference genomes have been generated for >20 cucurbit species, and variome and transcriptome profiling data have been rapidly accumulated for cucurbits. To efficiently mine, analyze and disseminate these large-scale datasets, we have developed an updated version of Cucurbit Genomics Database. The updated database, CuGenDBv2 (http://cucurbitgenomics.org/v2), currently hosts 34 reference genomes from 27 cucurbit species/subspecies belonging to 10 different genera. Protein-coding genes from these genomes have been comprehensively annotated by comparing their protein sequences to various public protein and domain databases. A novel 'Genotype' module has been implemented to facilitate mining and analysis of the functionally annotated variome data including SNPs and small indels from large-scale genome sequencing projects. An updated 'Expression' module has been developed to provide a comprehensive gene expression atlas for cucurbits. Furthermore, synteny blocks between any two and within each of the 34 genomes, representing a total of 595 pair-wise genome comparisons, have been identified and can be explored and visualized in the database.

Ubiquitination-mediated protein degradation plays important roles in ABA signal transduction and delivering responses to chloroplast stress signals in plants, but additional E3 ligases of protein ubiquitination remain to be identified to understand the complex signaling network. Here we reported that ZEITLUPE (ZTL), an F-box protein, negatively regulates abscisic acid (ABA) signaling during ABA-inhibited early seedling growth and ABA-induced stomatal closure in Arabidopsis thaliana. Using molecular biology and biochemistry approaches, we demonstrated that ZTL interacts with and ubiquitinates its substrate, CHLH/ABAR (Mg-chelatase H subunit/putative ABA receptor), to modulate CHLH stability via the 26S proteasome pathway. CHLH acts genetically downstream of ZTL in ABA and drought stress signaling. Interestingly, ABA conversely induces ZTL phosphorylation, and high levels of ABA also induce CHLH proteasomal degradation, implying that phosphorylated ZTL protein may enhance the affinity to CHLH, leading to the increased degradation of CHLH after ABA treatment. Taken together, our results revealed a possible mechanism of reciprocal regulation between ABA signaling and the circadian clock, which is thought to be essential for plant fitness and survival.

Fruit ripening is a highly complicated process, which is modulated by phytohormones, signal regulators and environmental factors playing in an intricate network that regulates ripening-related genes expression. Although transcriptomics is an effective tool to predict protein levels, protein abundances are also extensively affected by post-transcriptional and post-translational regulations. Here, we used RNA sequencing (RNA-seq) and tandem mass tag (TMT)-based quantitative proteomics to study the comprehensive mRNA and protein expression changes during fruit development and ripening in watermelon, a non-climacteric fruit. A total of 6,226 proteins were quantified, and the large number of quantitative proteins is comparable to proteomic studies in model organisms such as Oryza sativa L. and Arabidopsis. Base on our proteome methodology, integrative analysis of the transcriptome and proteome showed that the mRNA and protein levels were poorly correlated, and the correlation coefficients decreased during fruit ripening. Proteomic results showed that proteins involved in alternative splicing and the ubiquitin proteasome pathway were dynamically expressed during ripening. Furthermore, the spliceosome and proteasome were significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, suggesting that post-transcriptional and post-translational mechanisms might play important roles in regulation of fruit ripening-associated genes expression, which might account for the poor correlation between mRNAs and proteins during fruit ripening. Our comprehensive transcriptomic and proteomic data offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of fruit ripening.

Cucurbitaceae is one of the most genetically diverse plant families in the world. Many of them are important vegetables or medicinal plants and are widely distributed worldwide. The rapid development of sequencing technologies and bioinformatic algorithms has enabled the generation of genome sequences of numerous important Cucurbitaceae species. This has greatly facilitated research on gene identification, genome evolution, genetic variation, and molecular breeding of cucurbit crops. So far, genome sequences of 18 different cucurbit species belonging to tribes Benincaseae, Cucurbiteae, Sicyoeae, Momordiceae, and Siraitieae have been deciphered. This review summarizes the genome sequence information, evolutionary relationships, and functional genes associated with important agronomic traits (e.g. fruit quality). The progress of molecular breeding in cucurbit crops and prospects for future applications of Cucurbitaceae genome information are also discussed.

The mutation of ClZISO identified in EMS-induced watermelon leads to photosensitive flesh in watermelon. Watermelon (Citrullus lanatus) has a colorful flesh that attracts consumers and benefits human health. We developed an ethyl-methanesulfonate mutation library in red-fleshed line ‘302’ to create new flesh color lines and found a yellow-fleshed mutant which accumulated ζ-carotene. The initial yellow color of this mutant can be photobleached within 10 min under intense sunlight. A long-term light-emitting diode (LED) light treatment turned flesh color from yellow to pink. We identified this unique variation as photosensitive flesh mutant (‘psf’). Using bulked segregant analysis, we fine-mapped an EMS-induced G-A transversion in ‘psf’ which leads to a premature stop codon in 15-cis-ζ-carotene isomerase (ClZISO) gene. We detected that wild-type ClZISO is expressed in chromoplasts to catalyze the conversion of 9,15,9’-tri-cis-ζ-carotene to 9,9’-di-cis-ζ-carotene. The truncated ClZISOmu protein in psf lost this catalytic function. Light treatment can partially compensate ClZISOmu isomerase activity via photoisomerization in vitro and in vivo. Transcriptome analysis showed that most carotenoid biosynthesis genes in psf were downregulated. The dramatic increase of ABA content in flesh with fruit development was blocked in psf. This study explores the molecular mechanism of carotenoid biosynthesis in watermelon and provides a theoretical and technical basis for breeding different flesh color lines in watermelon.

Carotenoid pigments confer photoprotection and visual attraction and serve as precursors for many important signaling molecules.Herein,the orange-fruited phenotype of a tomato elite inbred line resulting from sharply reduced carotenoid levels and an increasedβ-carotene-to-lycopene ratio in fruit was shown to be controlled by a single recessive gene,oft3.BSA-Seq combined with fine mapping delimited the oft3 gene to a 71.23 kb interval on chromosome 4,including eight genes.Finally,the oft3 candidate gene SlIDI1,harboring a 116 bp deletion mutation,was identified by genome sequence analysis.Further functional complementation and CRISPR-Cas9 knockout experiments confirmed that SlIDI1 was the gene underlying the oft3 locus.qRT-PCR analysis revealed that the expression of SlIDI1 was highest in flowers and fruit and increased with fruit ripening or flower maturation.SlIDI1 simultaneously produced long and short transcripts by alternative transcription initiation and alternative splicing.Green fluorescent protein fusion expression revealed that the long isoform was mainly localized in plastids and that an N-terminal 59-amino acid extension sequence was responsible for plastid targeting.Short transcripts were identified in leaves and fruit by 5'RACE and in fruit by 3'RACE,which produced corresponding proteins lacking transit peptides and/or putative peroxisome targeting sequences,respectively.In SlIDI1 mutant fruit,SlBCH1 transcription involved in β-carotenoid catabolism was obviously suppressed,which may be responsible for the higherβ-carotene-to-lycopene ratio and suggested potential feedback regulatory mechanisms involved in carotenoid pathway flux.

为了探明低温弱光环境对西瓜果实糖分积累的调节机制,本试验通过分析棉籽糖水解酶碱性α-半乳糖苷酶基因aga2突变体转录组数据发现低温弱光下aga2突变体西瓜果实中有686个基因上调表达,990个基因下调表达,进一步通过GO和KEGG分析发现MYB、NAC、WRKY等转录因子上调表达抵御低温弱光;而肌醇半乳糖苷合酶(Cla009222)、α-半乳糖苷酶(Cla022883、Cla019238)等与糖分代谢相关的基因在低温弱光下下调表达可能是导致aga2突变体果实糖含量下降的主要原因.本试验通过分析转录组数据,挖掘响应低温弱光的西瓜糖分积累相关基因,为西瓜果实响应低温弱光的糖代谢调控网络奠定了基础.

How raffinose (Raf) family oligosaccharides, the major translocated sugars in the vascular bundle in cucurbits, are hydrolyzed and subsequently partitioned has not been fully elucidated. By performing reciprocal grafting of watermelon (Citrullus lanatus) fruits to branch stems, we observed that Raf was hydrolyzed in the fruit of cultivar watermelons but was backlogged in the fruit of wild ancestor species. Through a genome-wide association study, the alkaline alpha-galactosidase ClAGA2 was identified as the key factor controlling stachyose and Raf hydrolysis, and it was determined to be specifically expressed in the vascular bundle. Analysis of transgenic plants confirmed that ClAGA2 controls fruit Raf hydrolysis and reduces sugar content in fruits. Two single-nucleotide polymorphisms (SNPs) within the ClAGA2 promoter affect the recruitment of the transcription factor ClNF-YC2 (nuclear transcription factor Y subunit C) to regulate ClAGA2 expression. Moreover, this study demonstrates that C. lanatus Sugars Will Eventually Be Exported Transporter 3 (ClSWEET3) and Tonoplast Sugar Transporter (ClTST2) participate in plasma membrane sugar transport and sugar storage in fruit cell vacuoles, respectively. Knocking out ClAGA2, ClSWEET3, and ClTST2 affected fruit sugar accumulation. Genomic signatures indicate that the selection of ClAGA2, ClSWEET3, and ClTST2 for carbohydrate partitioning led to the derivation of modern sweet watermelon from non-sweet ancestors during domestication.

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in fruit ripening and quality. The watermelon genome encodes 80 NAC genes, and 21 of these NAC genes are highly expressed in both the flesh and vascular tissues. Among these genes, ClNAC68 expression was significantly higher in flesh than in rind. However, the intrinsic regulatory mechanism of ClNAC68 in fruit ripening and quality is still unknown. In this study, we found that ClNAC68 is a transcriptional repressor and that the repression domain is located in the C-terminus. Knockout of ClNAC68 by the CRISPR-Cas9 system decreased the soluble solid content and sucrose accumulation in mutant flesh. Development was delayed, germination was inhibited, and the IAA content was significantly decreased in mutant seeds. Transcriptome analysis showed that the invertase gene ClINV was the only gene involved in sucrose metabolism that was upregulated in mutant flesh, and expression of the indole-3-acetic acid-amido synthetase gene ClGH3.6 in the IAA signaling pathway was also induced in mutant seeds. EMSA and dual-luciferase assays showed that ClNAC68 directly bound to the promoters of ClINV and ClGH3.6 to repress their expression. These results indicated that ClNAC68 positively regulated sugar and IAA accumulation by repressing ClINV and ClGH3.6. Our findings provide new insights into the regulatory mechanisms by which NAC transcription factors affect fruit quality and seed development.

京颖是以自交系XM为母本,以自交系HX3为父本配制而成的早熟优质耐裂耐贮小型西瓜一代杂种.植株生长势较强,嫁接后低温条件下易坐瓜,果实发育期30～35 d(天),全生育期约90 d(天);果实椭圆形,果皮绿色覆墨绿色锯齿状窄条纹,单瓜质量约2.0 kg,果肉红色、均匀,中心糖含量12％～13％,最高可达15.7％;果皮薄而韧,耐裂,不易脱水,肉质脆嫩,口感佳,耐贮运,货架期可达15 d(天);每667 m2产量3500 kg左右,适于全国各地保护地小型西瓜高品质栽培及远距离运输使用.